The long-term goal of the proposed research is to understand the molecular mechanisms underlying visual transduction in the fruitfly, Drosophila melanogaster. Drosophila phototransduction is the fastest known G-protein coupled signaling cascade and it culminates in sodium and calcium influx, via the TRP, TRPL and TRPgamma cation channels. During the last few years, a large family of mammalian TRP-related proteins has been identified, which play critical roles in processes ranging from sensory physiology to vasorelaxation and male fertility. As is the case for the Drosophila TRPs, many of the mammalian TRP channels are activated through signaling pathways that are coupled to phospholipase C. However, the molecular mechanisms linking phospholipase C to the activity of the TRP channels are enigmatic. While the activation mechanisms of many mammalian TRP-related proteins have been addressed, a key limitation of most of these studies is that the analyses have focused exclusively on proteins overexpressed in heterologous systems. The goal of the current proposal is to characterize the mechanisms underlying the regulation of TRP channels using a multidisciplinary approach. Experiments are outlined to study the Drosophila TRP channels in parallel in vitro and in vivo systems, using a combination of molecular, genetic, biochemical, electrophysiological, calcium-imaging and cell biological approaches. The specific aims of the proposal are to test the following hypotheses: 1) TRP is regulated by PKC and PIP3; 2) TRP is regulated through direct interaction with a member of newly described class of membrane-associated proteins expressed exclusively in excitable cells; 3) TRPgamma is required for the visual response; and 4) TRP is a tetrameric channel with six transmembrane segments. The final specific aim (5) is devoted to characterizing a new activation mechanism underlying a mammalian TRP channel, TRPM1, and to initiate characterization of TRPM3. TRP channels appear to be important for human health as mutations in TRP-related channels cause a neurodegenerative disease, mucolipidosis, and polycystic kidney disease. Moreover, defects in TRP channels have been associated with changes in growth control and one TRP-related protein, TRPM1, may be a tumor suppressor.